Methylotrophic bacterium for the production of recombinant proteins and other products

ABSTRACT

The present invention relates to a method of producing a recombinant peptide, a recombinant protein, or a product from metabolic engineering using a genetically modified methylotrophic bacterium, and more particularly to  Methylobacterium extorquens  ATCC 55366. The method comprises introducing am expression vector into the methylotrophic bacterium, the expression vector comprising a polynucleotide sequence, coding for a peptide or protein, or allowing for production of a product from metabolic engineering under the control of a regulated promoter. the method also comprises growing the genectically modified methylotrophic bacterium in a minimal salts medium lacking organic sugars and containing methanol. A metal ion may be used for regulating the expression of the polynucleotide sequence by the promoter.

CROSS REFERENCE TO RLATED APPLICATIONS

The present application claim benefit under 35 U.S.C. §120 of earlierapplication Ser. No. 10/188,746 filed Jul. 5. 2002 and of applicationSer. No. 09/998,631 filed Dec. 3, 2001, both pending, the entire contentof both of which is hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to a system and method of producing recombinantpeptides or proteins and other products from metabolic engineering inprokaryotes. More specifically, the present invention relates to the useof the methylotrophic bacterium Methylobacterium extorquens ATCC 55366,in combination with novel expression vectors, as an efficient expressionsystem for recombinant peptides or proteins and industrially importantbulk chemicals.

BACKGROUND OF THE INVENTION

Current microbial processes for the production of recombinant proteinsuse either eukaryofic microorganisms (e.g. Pichia pastoris), which mayproduce unwanted glycosylation and other unwanted post-translationalmodifications, or prokaryotic cells. The bacterium Escherichia coli isthe best known and the most used prokaryotic expression system. The E.coli system, however, has drawbacks that include inclusion bodyformation (when undesired), high acetate production which tends toinhibit growth and product formation, and a requirement for relativelyexpensive carbon sources such as glucose.

Methylotrophic bacteria are a group of prokaryotic microorganisms thatcan utilize one-carbon (C₁) compounds more reduced than carbon dioxideas a source of carbon and energy. Formaldehyde, an intermediate in theoxidation of reduced C₁ compounds, is incorporated into cells carbon viathe serine pathway or via other pathways, and/or can be further oxidizedin a series of reactions to CO₂, generating energy in the form ofreducing equivalents.

Methylobacterium extorquens ATCC 55366 is a pink-pigmented faculativemethylotroph isolated from a hydrocarbon-contaminated sandy soil[Bourque et al (1992) Appl. Microbiol. Biotechnol. 37:7-12]. The growthof this bacterium in a fed-batch fermentation system developed byBourque et al [Bourque et al (1995) Appl. Microbiol. Biotechnol. 44(34):367-376] resulted in cultivation at very high cell densities using arelatively cheap substrate, methanol, for the production ofpoly-β-hydroxybutyrate, a very interesting polyester.

The ability to produce high biomass densities in fermenters, combinedwith the newly acquired genetic information obtained from the genomesequencing of M. extorquens AM1 [Alper (1999) Science 283:1625-1626],renders this microorganism extremely interesting as a potentialexpression system for recombinant peptides or proteins and for theproduction of industrially important bulk chemicals. In order to achievethese objectives, it is essential to identify efficient cloning vectorsand promoters for introducing new genes into M. extorquens.

It would be highly desirable to be provided with an efficient cloningvectors and promoters for introducing new genes into M. extorquens.

It would also be highly desirable to be provided with a method for theproduction of a large variety of products from metabolic engineering,which would help overcome some of the current problems.

SUMMARY OF THE INENTION

One aim of the present invention is to provide an efficient cloningvectors and promoters for introducing new genes into M. extorquens.

Another aim of the present invention is to provide a method for theproduction of products from metabolic engineering, which would helpovercome some of the problems faced with current microbial processes.

Such methods would include a new non-pathogenic prokaryotic microbialsystem, as an alternative to E. coli, for recombinant peptide or proteinexpression, which utilizes methanol as a source of carbon and energy forgrowth in chemically, protein-free, defined medium. Such methods coulduse a minimal culture medium combined with methanol as a carbon andenergy source, which would lower the costs of producing products frommetabolic engineering. Such systems would also include a new prokaryoticmicrobial system capable of producing recombinant peptides or proteinsand other products from metabolic engineering at levels comparable to P.pastoris (grams/liter) in a high cell density fermentation process frommethanol. Such methods would further include the development oftransformation vectors, which would be stably or satisfactorilymaintained in the cells in the presence of selective pressure such asantibiotics.

In accordance with one preferred embodiment of the present inventionthere is provided a method of producing a recombinant peptide, arecombinant protein or a product from metabolic engineering using agenetically modified methylotrophic bacterium under the control of aregulated promoter from a methylotrophic microorganism of the same ordifferent species as the methylotrophic bacterium, said methodcomprising the steps of:

a) introducing into said methylotrophic bacterium an expression vectorcomprising a polynucleotide sequence coding for a peptide or a proteinunder the control of a regulated promoter for producing said peptide orprotein or for allowing production of a product from metabolicengineering; and

b) growing said genetically modified methylotrophic bacterium in aminimal salts medium lacking organic sugars and containing methanol as asource of carbon and energy for said bacterium for a time sufficient toallow production of said peptide or protein or said product frommetabolic engineering.

In one embodiment, the method of the present invention further comprisesthe step of:

c) regulating expression of the polynucleotide sequence by the promoter.

In another embodiment of the present invention the regulated promoter isa metal regulated promoter and step c) is effected with a metal ion.

In another embodiment of the present invention the methylotrophicbacterium is of the species Methylobacterium.

In another embodiment of the present invention the methylotrophicbacterium is Methylobacterium extorquens ATCC 55366.

In one embodiment of the present invention, the polynucleotide sequenceis a gene coding for green fluorescent protein.

In another embodiment of the present invention, the polynucleotidesequence is a gene coding for an enzyme.

In another embodiment of the present invention, the polynucleotidesequence is a gene that encodes a peptide or protein that is not anenzyme.

In one embodiment of the present invention, the enzyme reads with acomponent within or from the culture medium to produce a peptide orprotein, or other product from metabolic engineering.

In another embodiment of the present invention, the peptide or protein,or other product from metabolic engineering, reacts with a componentwithin or from the culture medium to produce a product from metabolicengineering.

In one embodiment of the present invention, the polynucleotide sequenceis inserted into a vector suitable for introduction into amethylotrophic bacterium, wherein the vector is stably maintained withinthe methylotrophic bacterium during growth and replication of themethylotrophic bacterium in the presence of selection pressure, andwherein the vector allows for the expression of the polynucleotidesequence within the methylotrophic bacterium.

In one embodiment of the present invention, the selective pressure is anantibiotic.

In one embodiment of the present invention, the regulating expression ofthe polynucleotide sequence by the promoter is with Cu.

In one embodiment of the present invention, the promoter is the promoterpresent in the soluble methane monooxygenase (sMMO) operon ofMethylosinus trichosporium OB3b.

In another embodiment of the present invention, the promoter is pmxaFfrom a gene from a methylotrophic bacterium.

In a further embodiment of the present invention, the promoter is apromoter from a gene from a methylotrophic bacterium.

In another embodiment of the present invention, the promoter is apromoter from a gene from an organism other than a methylotrophicmicroorganism.

In one embodiment of the present invention, the expression vector ispmmoX-GFP-pRK310.

In another embodiment of the present invention, the expression vector ispmmoX-GFP-pVK101.

In another embodiment of the present invention, the expression vector ispLac-GFP-pJB3KmD.

In a further embodiment of the present invention, the expression vectoris pmxaF-GFP-pCM110.

In another embodiment of the present invention, the expression vector ispLac-GFP-pRK310.

In one embodiment of the present invention, the present invention can beused for high-throughput peptide or protein production, orhigh-throughput production of other products from metabolic engineering.

In another embodiment of the present invention, the present inventioncan be used for proteomics-based peptide or protein expression orproteomics-based expression of other products from metabolicengineering.

In one embodiment of the present invention, the growing the geneticallymodified methylotrophic bacterium is performed within a flask.

In another embodiment of the present invention, the growing thegenetically modified methylotrophic bacterium is performed within afermenter.

For the purpose of the present invention the following terms are definedbelow.

The term “methylotrophic bacterium” is intended to mean a group ofprokaryotic microorganisms that can utilize one-carbon (C₁) compoundsmore reduced than carbon dioxide as a source of carbon and energy.

The term “GFP” is intended to mean green fluorescent protein.

The term “expression vector” is intended to denote a DNA molecule,linear or circular, that comprises a segment encoding a polypeptide ofinterest operably linked to additional segments that provide for itstranscription. Such additional segments may include promoter andterminator sequences, and may optionally include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or may contain elements of both.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term “polynucleotide” denotes a single or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in A, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terns may describepolynucleotides that are single-stranded or double stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

The term “polypeptide” is intended to denote a polymer of amino acidresidues joined by peptide bonds, whether produced naturally orsynthetically, Polypeptides of less than about 10 amino acid residuesare commonly referred to as “peptides”.

The term “promoter” is intended to denote a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding regions of genes.

The term “facultative methylotroph” is intended to denote a bacteriumable but not obliged to grow in and perhaps use methanol as a carbonand/or energy source, but will also survive and perhaps grow in theabsence of methanol.

The term “obligate methylotroph” is intended to denote a bacteriumobliged to grow in and perhaps use methanol as a carbon and/r energysource, the bacterium will not survive or grow i the absence ofmethanol.

The term “and other products from metabolic engineering” is intended tomean, without limitation, plasmids for gene therapy or to support R&Dactivities, enzymes (cellulases, proteases, lipases), pigments (betacarotene, food colorants, antioxidants), vitamins (vitamin B12, biotin,riboflavin), amino acids (lysine, tryptophane, tyrosine, alanine),polysaccharides (pullulan, cellulose, chifin), biosurfactants(rhamnolipids, emulsan), biopestcides (Bt toxins, TMOF), hormones(insulin), antibiotics (tetracycline, penicillin, gramicidin,kanamycin). and biomaterials (silk, elastin, albumins).

BRIEF DESCRIPTION OF RHE DRAWINGS

FIG. 1 illustrates the schematic strategy used to create differentGFP-carrying plasmids.

FIG. 2 illustrates GFP production by M. extorquens in LB or CHOI media.

FIG. 3 illustrates GFP production by M. extorquens dependent on copperconcentration in the medium. Bars represent the error deviation withinfour independent fluorescence measurements.

FIG. 4 illustrates the production of GFP during the growth of M.extorquens (clone 3-63, pmmoX-GFP-pVK101 construct). Bars represent theerror deviation within four independent fluorescence measurements.

FIGS. 5A to 5C illustrate growth of Methylobacterium extorquens cloneM123A in 9-L fed-batch fermentation in the presence (●) or absence (▴)of tetracycline, expressed in terms of biomass yield (g cell dry weight(CDW)/L) (FIG. 5A), GFP production (mg protein/liter) (FIG. 5B) and GFPspecific yield (mg protein/g CDW) (FIG. 5C)

FIGS. 6A to 6C illustrate growth of Methylobacterium extorquens clone23-16 in 9-L fed-batch fermentation in the presence (●) or absence (▴)of tetracycline, expressed in terms of biomass yield (g cell dry weight(CDWYL) (FIG. 6A), GFP production (g protein/liter) (FIG. 6B) and GFPspecific yield (mg protein/g CDW) (FIG. 6C).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a new prokaryotic expressionsystem that can overcome drawbacks inherent in using current eukaryoticor prokaryotic cells for the production of recombinant peptides orproteins and other products from metabolic engineering. In particular,the present invention relates to the use of various expression vectorsthat can be used for recombinant peptide or protein expression andproduction of other products from metabolic engineering in M.extorquens. M. extorquens is a prokaryotc methylotrophic bacterium knownto lead to high biomass densities in fermenters and whose genome hasbeen completely sequenced. This microorganism is, therefore, extremelyattractive as a potential expression system. The present inventionprovides a new prokaryotic microbial system capable of producingrecombinant peptides or proteins and other products from metabolicengineering at high levels in a high cell density fermentation processfrom methanol.

Bacterial Strains, Plasmids and Growth Conditions

M. extorquens ATCC 55366 [Bourque et al. (1992) Appl. Microbiol.Biotechnol. 37:7-12] was grown as described previously [Bourque et al.(1995) Appl. Microbiol. Biotechnol. 44(304):367-376]. The ATCC number55366 is the number designated to the purified specimen culturedeposited on Oct. 14, 1992 with the American Type Culture Collection(ATCC, 12301 Parklawn Drive, Rockville, Md. 20852, United States ofAmerica). Electro-competent cells of M. extorquens were prepared by themethod of Toyama et al. [Toyama et al. (1998) FEMS Microbiol. Lett.166(1):1-7] after slight modifications. Cells were grown in CHOI medium(containing 1% v/v methanol) until the culture reached an OD₆₀₀≈0.6-0.8.Cells were harvested by centrifugation (1800×g, 10 min, 4° C.) andwashed twice with ice-cold sterile 10% (v/v) glycerol solution. The cellsuspension was concentrated 10-fold in 10% glycerol. dispensed in 400-μlaliquots and kept at −80° C. Electro-competent cells (100 μl) were mixedwith DNA solution (500 ng) in a 0.2-cm cuvette chilled on ice.Electroporation was carried out using a Gene Pulser (Bio-Rad) with thefollowing parameters: 2.5 kV, 400Ω, 25 μF, to a final field strength of12.5 kV cm⁻¹. After cells had been pulsed, 1 ml of ice-cold sterileLuria-Bertani low salts (LBLS) medium was immediately added to thecuvette, the cell suspension transferred into a test tube, and thenincubated at 30° C. for 24 h. Transformed clones were selected in LBnS(Luria-Bertani without NaCl) agar medium with appropriate antibiotics(kanamycin, 50 μg ml⁻¹; tetracycline, 20 μg ml⁻¹). Escherichia coli DH5α(Life Technologies Gibco BRL) or Top 10 (Invitrogen) for pCM constructswas cultivated at 37° C. in LBLS broth or on agar plates. Plasmids in E.coli were selected with ampicillin (100 μg ml⁻¹), kanamycin (50 μg ml⁻¹)or tetracycline (20 μg ml⁻¹), Information on the plasmids used for thepresent invention is given in Table 1. TABLE 1 Plasmids used PlasmidDescription Source pJB3KmD^(a) cloning vector, IacZ′, oriV, onT, Ap^(┌),[1] Km, 6.1 kb PRK310 cloning vector, IacZ′, oriV, onT, Tc^(┌), [2] 19kb PVK101 cloning vector, IacZ′, oriV, Tc^(┌), Km^(┌), [3] 20 kbPMTL1000 cloning vector, IacZ′, ori, Ap^(┌), [4] ˜12 kb PQBI63expression vector, 77, ori, Ap^(┌), [5] 6.3 kb PCM110 cloning vector,PmxaF, oriV, onT, [6] Tc^(┌), ˜5.8 kb GFP-pMTL1000 pMTL1000 with 0.78-kbinsert This containing GFP coding sequence invention from pQBI63PLac-GFP-pJB3KmD pKJ3KmD with 0.78-kb insert This containing GFP codingsequence invention from pQBI63 PLac-GFP-pRK310 pRK310 with ˜0.78-kbinsert This containing GFP coding sequence invention from pQBI63PmmoX-GFP- pRK310 with ˜9.5-kb insert This pRK310 containing sMMO andGFP coding invention sequence from GFP-pMTL1000 PmmoX-GFP- pVK101 with˜9.5-kb insert This pVK101 containing sMMO and GFP coding inventionsequence from GFP-pMTL1000 PmxaF-GFP-pCM110 PCM110 with ˜1-kb insertThis containing GFP coding sequence invention from pQBI63Ap^(┌), Km^(┌), Tc^(┌) denote resistance to ampicillin, kanamycin andtetracyclin, respectively.^(a)Accession Databank No. U75323.[1] Blatny et al. (1997) Appl. Environ. Microbiol. 63(2):370-379.[2] Toyama et al. (1998) FEMS Microbiol. Lett. 166-(1):1-7.[3] Knauf and Nester (1982) Plasmid 8:45-54.[4] Nielsen et al. (1997) Mol. Microbiol. 25(2):399-409.[5] Quantum Biotechnologies, Inc. (1998) Autofluorescent Proteins:Applications Manual. 11NO98.[6] Marx et al., (2001) Microbiology 147, 2065-2075.Construction of Plasmids

vitro DNA manipulation for cloning in E. coli was performed as describedby Sambrook et al. [Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.]. The strategy used to create different GFP carrying plasmids(except PmxaF-GFP-pCM110, see further below) is represented in FIG. 1.The set of primers used were: (a) GFP/BamH1.2 (5′-GAA TCG GGA TCC TCAGTT GTA CAG TTC ATC CAT GG-3′; BamHI restriction site underlined; SEQ IDNO:1) and RBS/Psti.2 (5′-AAC AAA CTG CAG AAT AAT TTT GTT TAA CTT TAA GAAGG-3′; Pstl restriction site underlined; SEQ ID NO:2); and (b) RBS/Mlul(5′-CAC GAC GCG TTG AAA TAA TTI TGT TTA ACT TTA AGA AGG-3′, Mlulrestriction site underlined; SEQ ID NO:3) and GFP/Xbal (5′-TGC TCT AGATCA GTT GTA CAG TTC ATC CAT GC-3′, Xbal restriction site underlined; SEQID NO:4). The polymerase chain reaction conditions in both cases were:hot start at 94° C. for 2 min and then 30 cycles of amplification (94°C., 30 s; 55° C., 30 s; 72° C., 30 s) followed by a final extension at72° C. for 10 min.

Detection of GFP Expression in M. extorquens

Selected clones of M. extorquens carrying GFP constructs were grown inLBnS or in CHOI medium containing 1% methanol and the appropriateantibiotic (kanamycin, 20 μg ml⁻¹; tetracycline, 20 μg ml⁻¹) at 30° C.,250 rpm. After 72 h of incubation, cells were harvested bycentrifugation and washed twice with sterile, deionized water. Cellswere resuspended in 700 μl of water and two 100 μl aliquots weredispensed into 96-well plates. The remaining 500 μl was used todetermine cell dry weight. Cells harboring pJB3KmD, pRK310, pVK101 orpCM110 were used as control, and their fluorescence was subtracted fromvalues obtained with cells harboring plasmids containing the gfp gene.

GFP production was determined in M. extorquens cells growing in CHOImedium. Cells were initially grown in 50 ml CHOI medium until themid-exponential phase (OD₆₀₀=0.8). A 2% inoculum was then used to startthe growth curve in 200 ml CHOI medium during which samples were takenfor measurement of OD₆₀₀, fluorescence and dry weight.

Fluorescence of cell suspensions was determined in a Cytofluor 2300System (Millipore) under excitation and emission wavelengths of 485 and530 nm, respectively.

The concentration of GFP was calculated based on a linear relationshipbetween concentration and fluorescence determined for solutions ofpurified GFP (Quantum Biotechnologies). GFP yield is reported as GFPconcentration (μg or mg as indicated) per unit of dry weight (g).

Determination of Cu Concentration in Solution

Cells grown in the presence of Cu were harvested by centrifugation andthe supernatant fluid was collected for Cu analysis. Concentrated H₂SO₄(93%) (0.5 ml) was added to 9.5 ml of supernatant fluid in order tomaintain the pH lower than 2.0 (thus preventing Cu precipitation) for Cuions analysis using inductively coupled plasma-atomic spectrometer(ICP-AS; Thermo Jarel Ash, Trace Scan). The result was corrected withthe appropriate dilution factor and referred to as the final soluble Cuconcentration. The total Cu concentration added to the medium atpreparation was determined likewise by adding 5% (v/v) concentratedH₂SO₄ to the medium and then analyzing using ICP-AS.

Efficiency of Transformation of M. extorquens by Electroporation

An essential step in achieving the expression and stability ofheterologous genes in methylotrophic hosts is through the use ofsuitable broad-host-range vectors. The plasmids used here, pJB3KmD,pRK310 and pVK101, are derived from broad-host-range vectors which weredeveloped for Gram-negative bacteria [Knauf et al. (1982) Plasmid8:45-54; Ditta et al. (1985) Plasmid 13:1349-153; Blatny et al. (1997)Appl. Environ. Microbiol. 63:370-379]. Table 2 shows the time constantvalues obtained, as well as the efficiency of transformation for thedifferent constructs used to transform M. extorquens. An overallimprovement was observed on the efficiency of transformation of eachplasmid in the following order; pJB3KmD<pRK310<pVK101. While the timeconstant values were within the range obtained by Toyama et al. [Toyamaet al. (1998) FEMS Microbiol. Lett. 166(1):1-7] when applying similarelectroporation conditions, the efficiency of transformation observed inthe present invention for pRK310 (˜10³ cells μg⁻¹ DNA) was at least twoorders of magnitude lower than that obtained by them. This value was,however, dose to that obtained by Ueda et al. [Ueda et al. (1991) Ann.N. Y. Acad. Sci. 646:99-105] when they electroporated M. extorquens withpLA2917. Such differences might be due to strain differences or todiverse conditions used for preparing the electro-competent cells, aswell as due to specific electroporation conditions such as the timeconstant produced after each energy discharge. Nonetheless, thetransformation efficiencies obtained in the present invention are highenough for practical use in genetic manipulation. The cosmid pVK100(which resembles pVK101 except for the presence of the cos factor inpVK100) was shown to be mobilized from E. coli strains into M.extorquens AMI (previously known as Pseudomonas sp. AM1) by conjugationat frequencies of 10⁻¹ to 10⁻² [Fulton et al. (1984) J. Bacteriol.160(2):718-723]; these frequency values were lower than the onesobserved in the present invention. There are very few reports in theliterature on the use of electroporation as a means of introducing DNAinto M. extorquens. Although conjugation has been the preferredtechnique for transforming methylotrophic bacteria, electroporation wasproven here to be a faster and less laborious technique. TABLE 2 Timeconstant produced and efficiency of transformation of M. extorquens byelectroporation Time constant Efficiency of transformation Plasmid (ms)′(cells pg⁻¹ DNA) pJB3KmD 8.9 1.2 × 10² pRK310 8.8 2.3 × 10³ pVK101 8.61.1 × 10⁴ PLac-GFP-pJB3KmD 8.7 0.8 × 10² PLac-GPP-pRK310 8.8 3.6 × 10³PmmoX-GFP-pRK310 8.7 0.7 × 10² PmmoX-GFP-pVK101 8.1 2.0 × 10³′Time of exposure of cells to the high field strength applied.GFP Production under the Control of the lacZ Promoter

The gfp gene used in the present invention originated from a modifiedconstruct of the wild-type GFP [Quantum Biotechnologies, Inc. (1998)Autofluorescent Proteins: Applications Manual. 11NO98]. Itstranscription was under the regulation of the lacZ promoter, of thesoluble monooxygenase gene duster promoter mmoX [Nielson et al. (1997)Mol. Microbiol. 25(2):399-409] or the methanol dehydrogenase promoter,pmxaF of M. extorquens AM1 (Marx et al., (2001) Microbiology 147,2065-2075). The lacZ promoter has been successfully used for theexpression in GFP by several bacteria [Bermudez et al. (1999) MethodsEnzymol. 302:285-295]. The fusion of LacZ regulative elements inconstructs containing mosquitocidal endotoxins gene (crylVB) fromBacillus thuringiensis led to a significant increase of crylVB geneexpression in the obligate methylotroph Methylobacillus flagellatum[Marchenko et al. (2000) J. Ind. Microbiol. Biotechnol. 24(1);14-18].However, the absence of the lacl gene gives rise to a constitutivephenotype and thus the lac promoter is induced constitutively evenwithout inducers [Park et al. (1999) J. Microbiol. Biotechnol.9(6)811-819].

The lac promoter was recognized by M. extorquens in the presentinvention, in accordance with previous findings [Toyama et al. (1998)FEMS Microbiol. Left. 166(1):1-7]. was also found to be constitutivelyinduced. An interesting observation arose from the comparison betweenthe fluorescence produced by clones of M. extorquens carrying the GFPgene in either pJB3KmD or pRK310 (FIG. 2). The use of the latter led toat least a 100-fold improvement in the amount of GFP produced by eachclone.

An important difference was also observed In the amount of GFP producedby clones growing in either LB or CHOI medium (FIG. 2). Independently ofthe construct used, an improvement of at least 30% in the yield of GFPwas obtained when cells grew in CHOI medium. The CHOI medium wasdescribed as the ideal medium to obtain high biomass titers of M.extorquens [Bourque et al. (1995) Appl. Microbiol. Biotechnol.44(3-4):367-376]. The hypothesis that nutritional limitations mayinterfere with the production of GFP or with its chromophore activity[Tsien (1998) Annu. Rev. Biochem. 67:509-544] should be furtherinvestigated in order to explain the significant difference in yields ofGFP depending on the medium used.

GFP Production under the Control of mmoX Promoter

In the present invention, M. extorquens was transformed with constructscontaining the gfp gene under the control of the mmoX promoter presentin the soluble methane monooxygenase (sMMO) operon of anothermethylotrophic bacterium, Methylosinus trichosporium OB3b. In thismicroorganism, sMMO catalyzes the oxidation of methane to methanol. Thetranscriptional regulation of the smmo gene is known to becopper-dependent; sMMO is expressed only under conditions in which thecopper-to-biomass ratio is low. This allows for a strict control of theexpression of the gene under its promoter by controlling theconcentration of Cu in the medium [Nielsen et al. (1997) Mol. Microbiol.25(2):399-409].

Transformed M. extorquens cells were grown in defined medium (CHOI) sothat the effect of varying the concentrations of Cu²⁺ on the productionof GFP could be determined (FIG. 3). was found that the promoter wasrecognized by M. extorquens, therefore, allowing for the expression ofGFP. The increase in the initial Cu²⁺ concentration (from 0 to 57 μM)did not interfere with cell growth (as observed by the final dryweight). However, expression of GFP by clones carrying bothpmmoX-GFP-pRK310 and pmmoX-GFP-pVK101 was not strongly controlled by thepresence of Cu in the medium and repression was observed to some extentwhen the initial concentation of Cu in the medium was increased up to 57μM.

Copper specification in the medium and its effect on the activity ofsMMO is also relevant to understanding smmo regulation. Morton et al.[Morton et al. (2000) Appl. Environ. Microbiol. 66(4):1730-1733] foundno detectable sMMO activity when >2.63 μM Cu g⁻¹ of protein was present.Moreover, different sMMO activities were observed, depending on the typeof Cu complex present, which could be explained by the inability ofcells to actively transport Cu complexes into the cells, thus reducingCu bioavailability. In the present invention, analysis of the soluble Cupresent in the medium at the end of the cell growth experiments revealedthat between 11 and 21% of the Cu added to medium was present in itsfree form (FIG. 3). The remainder Cu was probably precipitated asoxides, hydroxides or ligand complexes, or accumulated by the cells.While the effect of different species of Cu could affect the regulationof pmmoX, the final concentrations of free Cu in the medium should behigh enough to totally inhibit pmmoX expression. However, a reduction of41 and 33% in GFP production was observed when the final Cuconcentration was 2.4 and 3.9 μM Cu mg⁻¹ of biomass (for the clones 1-3and 3-63, respectively).

The results for growth of M. extorquens carrying the pmmoX-GFP-pVK101construct (in the absence of Cu) (FIG. 4) showed that the yield of GFPduring growth reached its maximum at mid-exponential phase (about 700 μgof GFP g⁻¹ of biomass) and decreased as the culture reached the earlystationary phase of growth (350 μg of GFP g⁻¹ of biomass at stationaryphase). Since the fluorescence of cells remained constant during thestationary phase, this suggested that the reduced GFP yield observed maybe due to cessation of GFP production during this phase. There could beseveral factors related to the growth conditions of M. extorquenscarrying pmmX-GFP-pVK101 (including O₂ or redox potential limitations,which are known to dramatically affect the maturation of GFP [Tsien(1998) Annu. Rev. Biochem. 67:509-544]) that could explain the apparentend or slowing down of GFP production during stationary phase.

GFP has now been used as a model heterologous protein in order toidentify suitable vectors as well as efficient promoters for M.extorquens. pRK310, pVK101 and PCM110 constructs containing Lac, mmoXand mxaF promoters are valuable expression systems for GFP and theexpression of other industrially more important, genes in this bacteriumshould now be more easily accomplished.

GFP Production under the Control of the mxaF Promoter

The strategy used to create pmxaF-GFP-pCM110 (L23) involved digestingthe plasmid pQBI63 (Quantum Biotechnologies) which contains gfp gene,with Clal+Xbal. The Xbal/RBS/GFP/Clal band was excised and purified fromagarose gel using the QIAEX™ II Agarose Gel Extraction kit (QiagenInc.). Plasmid pCM110 [Marx, C. J. and M. E. Lidstrom. (2001)Microbiology 147, 2065-2075] was digested with Clal+Spel and theSpel/pCM110/Clal band was purified as previously. Ligation of bothfragments was performed overnight at 16° C. to give pmxaF-GFP-pCM110.

Clones M123A and 28-163 (harbouring the constructs pLac-GFP-pRK310 andpmxaF-GFP-pCM110, respectively) were grown for 24 h in Erlenmeyer flaskscontaining tetracycline, as described earlier. An aliquot of the culturegrowth was then used to inoculate flasks containing fresh medium withouttetracycline (5% inoculum) for another 24-h period. This procedure wasrepeated for 9 consecutive batches. A control set of experimentsconsisted of growing the clone in the same conditions, except thattetracycline was always added to the medium. For each batch, GFPconcentration and cell dry weight were measured and specific GFPproduction was determined as previously described.

Recombinant M. extorquens fed-batch cultures were performed using a 20-Lcontinuously stirred baffled fermenter (Chernap, Volkestwill,Switzerland) equipped with pH and pO₂ electrodes (Ingold), a foamsensor, and a mechanical foam breaker. For agitation, the biorector wasequipped with 3 Rushton impellers. The dissolved oxygen level wascontrolled around 15% of saturation by first, increasing agitation speedfrom 500 rpm to 1,000 rpm and then, by increasing the airflow supplyfrom 7 L/min to 15 L/min. All fed-batch bioreactor experiments wereconducted at pH 7.0 and 30° C. Ammonia solution (30%) was used as bothpH regulator and nitrogen source, and was added as needed in allfermentations. Chemical antifoam Ucarferm™ (Union Carbide, Dandury,Conn., U.S.A.) was used as needed to control excessive foaming.Fermenter containing 9 L of modified medium CHOI, and 20 mg ml⁻¹tetracycline in some experiments (see table 4 below), was inoculatedusing 1-L culture grown in flasks with or without antibiotic dependingon the experiment (see table 4 below).

On-line measurement of the methanol concentration in the culture mediumwas performed using a silicone membrane probe (Bioengineering Inc.)coupled with a semiconductor gas sensor [Bourque, D., et al., (1995)Appl. Microbiol. Biotechnol. 44(3-4), 367-376]. The methanolconcentration was kept around 0.05% (v/v) by using an on-off controllerfor the first 24-h period, and then a PID controller for the rest of thefermentation. Methanol was added using a variable-speed peristalticpump. Off-gas measurements were performed for O₂ (Servomex Paramagneticanalyzer) and CO₂ (Servomex Infrared analyzer) concentrations.

Fluorescence of cell suspensions derived from the fermenters, dispenseddirectly or after dilutions with phosphate buffered saline (pH 7.4) in96-well black polystyrene plats, was determined in a SPECTRAFluor™ Plus(TECAN Austria Gmbh, Grodlg, Austria) under excitation and emissionwavelenghts of 485 and 530 nm, respectively. Cell dry weight for thosesamples was measured using a Moisture Analyzer MA 30 (Sartorius,Canada).

Off-line measurement of methanol concentrations were determined by gaschromatography (GC) using a Varian gas chromatograph CP-3800 (VarianAnalytical Instruments, Walnut Creek, Calif., U.S.A.) equipped with aflame ionisation detector and a DB™-5 capillary column (5% phenyl and95% methyl silicone, 25 m×0.20 mm×0.33 μm; Alltech, Guelph, Ont.,Canada). Other conditions were: column temperature, 70° C. for 2 min,70-105° C. for 3 min and 105° C. for 0.5 min; injector and detectortemperatures, 250° C. and 275° C. respectively; carrier gas He at flowrate of 30 m/min.

The feasibility of using microbial heterologous expression systems inindustrial scale depends, among other factors, on how stable therecombinant DNA is within the host cell. For the purpose of simplifyingthe purification of the recombinant product, it is desirable that cellsare able to grow in fermenters in the absence of selective pressure suchas antibiotics. This can be achieved by using plasmids that are kept inthe cells without modifications in their nucleotide sequence, even ifthey are grown for several generations in the absence of antibiotics. Inorder to establish the plasmid stability—as determined by the number ofgenerations—of M. extorquens clones M123A and 28-163 (harbouring theconstructs pLacGFP-pRK310 and pmxaF-GFP-pCM110, respectively), repeatedsequential batch cultures were carried out in the absence oftetracycilne. A 5% v/v sample of the culture was used as a pre-inoculumin fresh medium and incubated for equivalent amount of time. The processwas repeated for 9 consecutive batches. While the production of GFP waskept constant for as long as the cells were grown in tetracycline,relative reductions (based on the specific yields of the first batchculture) of 89% and 99% on GFP production was observed for the clonesM123A and 28-163 respectively, after 45 generation times in the absenceof antibiotic (Table 3). After 15 generations, corresponding to thenumber of generations obtained in a 65-h fed-batch culture, the specificGFP production decreased 40% and 35%. respectively. These resultsindicate that the addition tetracycline is desirable to maintain themaximum recombinant protein production in the conditions applied in theexperiments. Depending on the nature and potential intrinsic value ofthe recombinant product, however, the application of such a system atreduced productivity conditions could still be economically justified,as long as the production process consists of batch fermentations. TABLE3 Specific GFP yield (mg protein g cell dry weight (CDW)) by clonesM123A and 23-16 growing in repeated sequential batch cultures, inpresence or absence of tetracycline Specific GFP production (mg/g) ±standard deviation # of Clone 23-16 Batch gen- Clone M123A Without cul-era- Without With tetra- With ture tions tetracycline tetracyclinecycline tetracycline 1  5 0.49 ± 0.04 0.20 ± 0.06 48 ± 5  40 ± 6 2 100.16 ± 0.06 0.3 ± 0.2 33 ± 3  35 ± 5 3 15 0.18 ± 0.01 0.45 ± 0.05 17 ±2  32 ± 4 4 20 0.12 ± 0.02 0.33 ± 0.01 19 ± 2  36 ± 3 5 25 0.12 ± 0.010.06 ± 0.04 12 ± 2  33 ± 3 6 30 0.2 ± 0.1 0.22 ± 0.02 3.0 ± 0.5 32 ± 2 735 0.14 ± 0.06 0.25 ± 0.03 2.7 ± 0.9 43 ± 5 8 40 0.05 ± 0.01 0.349 ±0.002 1.5 ± 0.6 38 ± 7 9 45 0.054 ± 0.005 0.26 ± 0.03 0.3 ± 0.2 37 ± 9

Although shake flask experiments may be useful in determining expressionlevels of recombinant proteins by recombinant cells (fed-batchcultures), these expression levels may not be comparable to the levelsobtained under fed-batch conditions. Changes in growth parameters suchas dissolved oxygen, substrate availability and agitation rate can havea profound effect on the expression levels of recombinant proteins[Glick, B. R. (1995) Biotechnol. Adv. 13(2), 247-261]. Therefore, it isnecessary to ascertain the effect of scale up of M. extorquens infed-batch fermentations on the expression levels of recombinantproteins. The following section discusses the performance of M.extorquens as a heterologous system when grown to high cell densities infed-batch fermenters.

FIG. 5A depicts the growth of M. extorquens done M123A harbouring mepLac-GFP-pRK310 construct in a 20-L fed-batch bioreactor, in thepresence or absence of tetracycline. The total biomass production andgrowth rates (represented by calculated μ_(max), in Table 4) in bothconditions were similar. After a 20-h incubation period, cells in thefermenter entered the exponential growth phase, which lasted for atleast the next 40 hours. However, the GFP production was markedlyreduced when cells were grown in the absence of antibiotics (FIG. 5B),leading to a total specific GFP yield of up to 50% less than thatobserved for cells growing in medium containing tetracycline (FIG. 5Cand Table 4). The reduction levels were comparable to that obtained inflask batches for this clone: after 15 generations, about 40% of theproduction capacity of the clone is lost by the lack of selectivepressure in the medium. These results indicate that there may be one ora combination of factors that affect the recombinant protein synthesis.Among them, the metabolic load, or changes in the metabolism of the hostmicroorganism in ways that may impair the organism's normal metabolicfunction [Glick, B. R. (1995) Biotechnol. Adv. 13(2), 247-261]. A greatamount of energy may be required to maintain the presence of the newlyintroduced plasmid DNA in a host cell and to produce/overproduce aforeign (recombinant) protein. This may lead to plasmid segregationalinstability in the absence of selective pressure. When this occurs, theplasmid-less cells out compete the plasmid-bearing cells, resulting inthe ultimate decrease in the final yield of recombinant protein [Glick,B. R. (1995) Biotechnol. Adv. 13(2), 247-261; and Zabrsde, D. W. and E.J. Arcud. (1986) Enzyme Microb. Technol. 8, 706-716,4]. Other factorssuch as oscillations in the carbon source concentration [Lin, H. Y. andP. Neubauer. (2000) J. Biotechnol. 79, 27-37] and oxygen fluctuations[Namdev, P. K., et al., (1993) Biotechnol. Bioeng. 41, 666-670] couldalso interfere in both plasmid stability and recombinant proteinproduction. TABLE 4 Final recombinant protein (GFP) and biomass yieldsfor recombinant clones M123A and 23-16 grown either in presence (+) orabsence (−) of selective pressure in fed-batch fermentations Yield¹Strain/ Biomass GFP/ Fermen- Selective Length X μ_(max) GFP/X Methanoltation pressure (h) (g/L) (h⁻⁷) (mg/g) (mg/g) M123A 1 + 73.5 72.1 0.1830.8 0.2 3 − 66 54.1 0.185 0.5 0.1 5 − 66 66.2 0.185 0.4 0.1 23-16 6 + 6560.0 0.177 42.6 11.5 7 − 65 60.4 0.160 40.3 14.7 8* − 47 54.6 0.164 67.717.4*fermentation with the addition of enriched air with pure oxygen;¹production yields are relative to the biomass or total methanolconsumed;²Maximum growth rate (μmax) is calculated based on the OTR (oxygentransfer rate)

Clones M123A and 23-16 (harbouring pmxaF-GFP-pCM110) possessed similarfed-batch growth characteristics in 20-liter fermenters regardless ofthe presence or absence of tetracycline (FIGS. 5A and 6A). However, thelevel of recombinant protein (GFP) production differed significantly.Clone 23-16 produced sixty orders of magnitude more recombinant GFP thanclone M123A (FIGS. 5B and 6B; note that y axis units are in mg/L andg/L, respectively), reaching 3 g GFP/L. This higher productivity isrelated to the presence of a natural promoter to M. extorquens, pmxaF,which controls the expression of the enzyme methanol dehydrogenase in M.extorquens AM1 [Marx, C. J. and M. E. Lidstrom. (2001) Microbiology 147,2065-2075]. This strong promoter is always “turned on” during themicrobial growth due to the constant addition of methanol as the carbonsource.

Although growth rates for the clone 23-16 were in general lower thanthose for the M123A, and slightly lower in experiments run in theabsence of antibiotics, GFP production appeared to be unaffected by thepresence or absence of selective pressure (FIG. 6B). This suggests thatthe construct pmxcaF-GFP-pCM110 is indeed more stable than thepLac-GFP-pRK310 construct in M. extorquens cells. The fact is confirmedby the total GFP production yield obtained at the end of thefermentation in the absence of antibiotic (fermentation #7, Table 4),which was very similar to that in the presence of antibiotic(fermentation #6). These results contrast, however, with those obtainedin flask experiments where after approximately 15 generations, about 35%of the GFP yield is lost when cells grow in the absence of antibiotics.This difference could be due to very different growth conditions infed-batch fermentations that do not reflect those of batch systems,emphasizing the need for scale-up optimization. is well known that thenumber of generations achieved in the fermenter, as well as temperatureand pH conditions, agitation, aeration and pressure are factors thataffect bioprocesses by scale [Thiry, M. and D. Cingolani. (2002) TrendsBiotechnol. 20(3), 103-105].

Mass transfer limitations in the fermenter can result in growthlimitations derived from irregular distribution of oxygen to cells. Thisis particularly true in fed-batch processes, where dissolved oxygen isoften a limiting factor if a high growth rate is reached [Thiry, M. andD. Cingolani. (2002) Trends Biotechnol. 20(3), 103-105]. One way ofovercoming this problem is by enriching air with pure oxygen, thereforeincreasing the dissolved oxygen supply without significant increasingthe dissolved carbon. Experiment #8 was carried out with this approachas the objective. Indeed, GFP yields were increased considerably (68%higher than when non-enriched air was used, experiment #7), despite thesimilar maximum growth rate obtained (Table 4). The total amount ofbiomass obtained was about 10% lower in this condition, but the rate ofGFP produced per methanol consumed (17.4 mg GFP/g MeOH) was the highestfor all the experiments run, indicating that the metabolism of cells wasshifted to producing more GFP for the same amount of MeOH added. Thissuggests that dissolved oxygen rate in the bioreactor might be animportant controlling step in the production of recombinant protein infed-batch fermentations. The limited amount of dissolved oxygen in thegrowth medium is often insufficient for both optimal host cellmetabolism maintenance and expression. In experiments with E. coli,whenever the dissolved oxygen was rapidly reduced the number ofplasmid-containing cells decreased to less than 1% of the cellpopulation, even when the selective pressure was present If oxygenlimitations exist even in a transient state throughout the culture, theforeign plasmid may become unstable and lost from the recombinant cells,thereby reducing the yield of the recombinant protein [Glick, B. R.(1995) Biotechnol. Adv. 13(2), 247-261; and White, M. D., et al., (1994)Bacterial, yeast and fungal cultures: the effect of microorganism typeand culture characteristics on bioreactor design and operation. In:Bioreactor System Design (Asenjo, J. A. and J. Merchuk Eds.)1-34. MarcelDekker, N.Y.].

An esterase (estl) gene of Lactobacillus casel was also successfullyover-expressed in Methylobacterium extorquens using its homologousmethanol dehydrogenase promoter (PmxaF), and the transformed cellsproduced the enzyme in its active form.

A high-cell density fed-batch fermentation containing methanol a solesource of carbon and energy was carried out under optimal conditions ofdissolved oxygen, pH and temperature. Recombinant M. extorquenscontaining pCEST was grown at pH 6.8 and 30° C. yielding a cell densityof 42 g dry cell weight/L after 48 h of growth. Production ofrecombinant esterase was observed at 20 h of growth and continued toincrease until cell harvest (50 h). The total esterase yield obtained(75.2×10³ units/ml) is 980-fold higher than in wild type L. casel, and 2to 2.5 times higher than in the E. coli. The recombinant enzymes werepurified to homogeneity by a single purification step resulting in highyields. No significant differences in physicochemical and catalyticproperties were observed between the recombinant enzyme and the nativeenzymes.

The classic large-scale fermentation process today is a fed-batchprocess with high final density based on as cheap a substrate aspossible. The advantages of this type of bioreactor over the batchsystem include the prevention of overflow metabolism and minimization ofinhibition effects caused by the main carbon source being added in highquantifies at the start of the fermentation [Liden, G. (2002) BioprocessBiosyst. Eng. 24, 2732791. While oxygen and nutrient transferlimitations can be of concern when high cell densities are achieved inthe fermenter, with increasing chances of segregational plasmidinstability, this can be partially overcome by changing operationalparameters change such as the supply of pure oxygen to the cultures, asshown in the present application.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. A method of producing a recombinant peptide, a recombinant protein ora product from metabolic engineering using a genetically modifiedmethylotrophic bacterium under the control of a regulated promoter froma methylotrophic microorganism of the same or different species as themethylotrophic bacterium, said method comprising the steps of: a)introducing into said methylotrophic bacterium an expression vectorcomprising a polynucleotide sequence, coding for a peptide or a proteinunder the control of a regulated promoter for allowing production ofsaid peptide or protein or for allowing production of a product frommetabolic engineering; and b) growing said genetically modifiedmethylotrophic bacterium in a minimal salts medium lacking organicsugars and containing methanol as a source of carbon and energy for saidbacterium for a time sufficient to allow production of said peptide orprotein or said product from metabolic engineering.
 2. The method ofclaim 1 further comprising the step of: c) regulating expression of saidpolynucleotide sequence by said promoter.
 3. The method of claim 1,wherein said regulated promoter is a metal regulated promoter.
 4. Themethod of claim 2, wherein step c) is effected with a metal ion.
 5. Themethod of any one of claims 1, wherein step b) is effected in anoxygen-enriched medium.
 6. The method of claim 5 wherein theoxygen-enriched medium is an oxygen-enriched CHOI medium.
 7. The methodof any one of claims 1, wherein said methylotrophic bacterium is afacultative methylotroph or an obligate methylotroph.
 8. The method ofany one of claims 1, wherein said methylotrophic bacterium is of thespecies Methylobacterium.
 9. The method of any one of claims 1, whereinsaid first methylotrophic microorganism is Methylobacterium extorquensATCC
 55366. 10. The method of any one of claims 1, wherein saidpolynucleotide sequence is a gene coding for green fluorescent protein.11. The method of any one of claims 1, wherein said polynucleotidesequence is a gene coding for an enzyme.
 12. The method of claim 11,wherein said enzyme reacts with a component within or from said culturemedium to produce a biomaterial or a product from metabolic engineering.13. The method of any one of claims 1, wherein said peptide or proteinor said product from metabolic engineering reacts with a componentwithin or from said culture medium to produce a biomaterial.
 14. Themethod of any one of claim 1, wherein said vector is capable ofreproduction within said bacterium and said vector is stably maintainedwithin said bacterium during growth and replication of said bacterium,in presence of selective pressure.
 15. The method of claim 14, whereinsaid selective pressure is an antibiotic.
 16. The method of claim 1,wherein said vector allows for the expression of said polynucleotidesequence within said methylotrophic bacterium.
 17. The method of claim4, wherein said metal ion is Cu²⁺.
 18. The method of any one of claims1, wherein said promoter is the promoter present in the soluble methanemonooxygenase (sMMO) operon of Methylosinus trichosporium OB3b.
 19. Themethod of claim 1, wherein said promoter is pmxaF.
 20. The method ofclaim 1, wherein said vector is pmxaF-GFP-pCM110.
 21. The method of anyone of claims 1, wherein said vector is pmmoX-GFP-pRK310.
 22. The methodof any one of claims 1, wherein said vector is pmmoX-GFP-pVK101.
 23. Themethod of claim 2, further comprising the step of: d) controlling theexpression of said polynucleotide sequence with a promoter from a genefrom an organism other than a methylotrophic bacterium.
 24. The methodof claim 23, wherein said vector is pLac-GFP-pJB3KmD.
 25. The method ofclaim 23, wherein said vector is pLac-GFP-pRK310.
 26. The method of anyone of claims 1, wherein the use can be for high-throughput productionof a peptide, protein or product from metabolic engineering.
 27. Themethod of any one of claims 1, wherein the use can be forproteomics-based peptide or protein expression.
 28. The method of anyone of claims 1, wherein the step of growing said genetically modifiedmethylotrophic bacterium is performed within a flask or fermenter. 29.The method of any one of claims 1, wherein said protein is a polypeptidehaving >10 amino acid residues in length.
 30. The method of any one ofclaims 1, wherein said peptide is ≦10 amino acid residues in length. 31.An expression vector for producing a recombinant peptide, a recombinantprotein or a product from metabolic engineering In a methylotrophicbacterium, wherein said expression vector comprises a polynucleotidesequence coding for a peptide or a protein or allowing production of aproduct from metabolic engineering, under the control of a metalregulated promoter.
 32. An expression vector for producing a recombinantpeptide, a recombinant protein or a product from metabolic engineeringin a methylotrophic bacterium grown in a minimal salts medium lackingorganic sugars and containing methanol, wherein said expression vectorcomprises a polynucleotide sequence coding for a peptide or a protein orallowing production of a product from metabolic engineering, under thecontrol of a methylotrophic bacterium promoter.